Description
Background information
The MACSelect 4 – Transfected Cell Selection Kit contains the vectors pMACS 4-IRES.II and pMACS 4.1, a control vector, MACSelect 4 MicroBeads, CD4-FITC antibody for detection of cells before enrichment, MACSelect Control FITC antibody for detection of cells after enrichment, and a CD14-FITC antibody for analysis of the control experiment when doing a co-transfection. All components are also available separately. The pMACS 4-IRES.II vector enables the bicistronic expression of the gene of interest from a CMV promotor and the truncated CD4 molecule via an internal ribosome entry side (IRES). The pMACS 4.1 vector encodes the truncated CD4 marker and is recommended for co-transfection in combination with an expression vector encoding the gene of interest. Transfected cells are magnetically labeled with CD4 MicroBeads and subsequently separated from non-transfected cells.
Note: The CD4 marker is of human origin and sensitive to trypsin treatment. Therefore, the MACSelect Kk or MACSelect LNGFR Systems should be used instead of the MACSelect 4 System when working with human cells or cells which require trypsin for detachment.
Detailed procedure
Cells are transfected with the transfection method of choice using either the pMACS 4-IRES.II vector harboring the gene of interest for single vector transfection or with the pMACS 4.1 vector and an expression vector encoding the gene of interest. The time of optimal marker expression, which determines the incubation time post transfection, has to be taken into consideration for each individual experiment. The level of marker expression can be determined by staining an aliquot of transfected cells with the CD4-FITC antibody followed by fluorescence microscopy or flow cytometric analysis. Depending on the cell type, transfection method, and culture conditions cells generally need to be cultured between 6 and 84 h. After incubation, cells are harvested and magnetically labeled using MACSelect 4 MicroBeads. Cells are loaded onto an MS or LS Column, placed in a MiniMACS or MidiMACS Separator. After washing, only the magnetically labeled transfected cells are retained on the column. Magnetic separation can also be carried out on the autoMACS Pro Separator if automatic processing is required. After enrichment, cells can be analyzed by fluorescence microscopy or flow cytometry using the supplied MACSelect Control FITC Antibody.
Applications
The MACSelect 4 System facilitates the enrichment of a homogeneous cell population of transiently transfected cells eliminating the need for setting up stable cell lines. As the MACSelect 4 MicroBeads are non-toxic, the enriched cells can be directly cultured. Stable cell lines can also be rapidly and efficiently established by repeated magnetic enrichment without the need for toxic antibiotic treatment. The system can be used for the enrichment of transiently transfected or transduced primary cells to a purity that enables sensitive downstream analysis. It is suitable for transfected cell selection when a rapid, non-toxic and non-immunogenic method is required.
Downstream applications
After enrichment the transfected cells can be subjected to flow cytometry, fluorescence microscopy, (co-)immunoprecipitation experiments, Western blot analysis, or reporter gene assays in functional gene and drug screening, signal transduction, protein interaction, and RNAi knockdown studies.
Overview
The MACSelect 4 System uses the transiently expressed truncated human CD4 molecule as a surface marker to select transfected cells. It can be used in virtually all CD4- adherent or suspension cell lines and primary cells in combination with common transfection methods.
